High sensitivity assays for angiotensin converting enzyme

Abstract

A method for measuring the activity of angiotensin converting enzyme is disclosed. In this assay, benzoylphenylalanylalanylproline or a radioisotope labelled form thereof is used as the substrate for the angiotensin converting enzyme.

What is claimed is: 
     
       1. A method for measuring the activity of angiotensin converting enzyme in a sample of clinical material comprising: providing a reaction buffer comprising chloride ions in the range from 0.01 M to 0.5 M,   mixing the sample of clinical material together with benzoylPheAlaPro in the reaction buffer, the benzoylPheAlaPro having a final concentration within the range from 1×10 -10  M to 7×10 -5  M, to provide a reaction mixture,   incubating the reaction mixture in order to permit any angiotensin converting enzyme catalyzed reaction to proceed to a measureable extent,   stopping the reaction by acidifying to the reaction mixture, and   measuring the amount of remnant reaction product produced by angiotensin converting enzyme catalyzed hydrolysis of benzoylPheAlaPro.   
     
     
       2. A method according to claim 1 wherein at least a portion of the benzoylPheAlaPro molecules are radioactively labeled in the benzoylPhe moiety and the amount of radioactivity in any benzoylPhe produced by angiotensin converting enzyme catalyzed hydrolysis of benzoylPheAlaPro is measured, in order to measure the activity of any angiotensin converting enzyme in the sample of clinical material. 
     
     
       3. A method according to claim 1 wherein the benzoylPheAlaPro concentration is in the range from 1×10 -9  M to 7×10 -5  M, the reaction mixture contains chloride ions in the range from 0.01 M to 0.5 M, at least a portion of the benzoylPheAlaPro molecules are radioactively labeled in the benzoylPhe moiety, the remnant reaction product of any angiotensin converting enzyme catalyzed hydrolysis of benzoylPheAlaPro is separated from the dipeptide reaction product and any unhydrolyzed benzoylPheAlaPro by extracting the reaction mixture with toluene, and measuring the radioactivity of any benzoylPhe extracted by the toluene, in order to measure the amount of angiotensin converting enzyme catalyzed benzoylPheAlaPro hydrolysis, thereby measuring the activity of angiotensin converting enzyme in the sample of clinical material.